1. Field of the Invention
The present invention concerns polynucleic acids isolated from a porcine reproductive and respiratory syndrome virus (PRRSV), a protein and/or a polypeptide encoded by the polynucleic acids, a vaccine which protects pigs from a PRRSV based on the protein or polynucleic acids, methods of making the proteins, polypeptides and polynucleic acids, a method of protecting a pig from PRRS using the vaccine, a method of producing the vaccine, a method of treating a pig infected by or exposed to a PRRSV, and a method of detecting a PRRSV.
2. Discussion of the Background
Porcine reproductive and respiratory syndrome (PRRS), a new and severe disease in swine, was first reported in the U.S.A. in 1987, and was rapidly recognized in many western European countries (reviewed by Goyal, J. Vet. Diagn. Invest., 1993, 5:656-664; and in U.S. application Ser. No. 08/131,625, now U.S. Pat. No. 5,695,766, and Ser. No. 08/301,435). The disease is characterized by reproductive failure in sows and gilts, pneumonia in young growing pigs, and an increase in preweaning mortality (Wensvoort et al., Vet. Q., 13:121-130, 1991; Christianson et al., 1992, Am. J. Vet. Res. 53:485-488; U.S. application Ser. No. 08/131,625, now U.S. Pat. No. 5,695,766, and Ser. No. 08/301,435).
The causative agent of PRRS, porcine reproductive and respiratory syndrome virus (PRRSV), was identified first in Europe and then in the U.S.A. (Collins et al., 1992, J. Vet. Diagn. Invest., 4:117-126). The European strain of PRRSV, designated as Lelystad virus (LV), has been cloned and sequenced (Meulenberg et al., 1993, Virology, 192:62-72 and J. Gen. Virol., 74:1697-1701; Conzelmann et al., 1993, Virology, 193:329-339).
PRRSV was provisionally classified in the proposed new virus family of Arteriviridae, which includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus (SHFV) (Plagemann and Moennig, 1992, Adv. Virus. Res., 41:99-192; Godeny et al., 1993, Virology, 194:585-596; U.S. application Ser. No. 08/131,625, now U.S. Pat. No. 5,695,766, and Ser. No. 08/301,435). This group of single plus-strand RNA viruses shares many characteristics such as genome organization, replication strategy, morphology and macrophage tropism (Meulenberg et al., 1993; U.S. application Ser. No. 08/131,625, now U.S. Pat. No. 5,695,766, and Ser. No. 08/301,435). Subclinical infections and persistent viremia with concurrent antibody production are also characteristic histopathologic properties of the arteriviruses.
Antigenic, genetic and pathogenic variations have been reported among PRRSV isolates (Wensvoort et al., 1992, J. Vet. Diagn. Invest., 4:134-138; Mardassi et al., 1994, J. Gen. Virol., 75:681-685; U.S. application Ser. No. 08/131,625, now U.S. Pat. No. 5,695,766, and Ser. No. 08/301,435). Furthermore, U.S. and European PRRSV represent two distinct genotypes (U.S. application Ser. No. 08/131,625, now U.S. Pat. No. 5,695,766, and Ser. No. 08/301,435). Antigenic variability also exists among different North American isolates as well (Wensvoort et al., 1992). Marked differences in pathogenicity have been demonstrated not only between U.S. and European isolates, but also among different U.S. isolates (U.S. application Ser. No. 08/131,625, now U.S. Pat. No. 5,695,766, and Ser. No. 08/301,435).
The genomic organization of arteriviruses resembles coronaviruses and toroviruses in that their replication involves the formation of a 3xe2x80x2-coterminal nested set of subgenomic mRNAs (sg mRNAs) (Chen et al., 1993, J. Gen. Virol. 74:643-660; Den Boon et al., 1990, J. Virol., 65:2910-2920; De Vries et al., 1990, Nucleic Acids Res., 18:3241-3247; Kuo et al., 1991, J. Virol., 65:5118-5123; Kuo et al., 1992; U.S. application Ser. No. 08/131,625, now U.S. Pat. No. 5,695,766, and Ser. No. 08/301,435). Partial sequences of several North American isolates have also been determined (U.S. application Ser. No. 08/131,625, now U.S. Pat. No. 5,695,766, and Ser. No. 08/301,435; Mardassi et al., 1994, J. Gen. Virol., 75:681-685).
The genome of PRRSV is polyadenylated, about 15 kb in length and contains eight open reading frames (ORFs; Meulenberg et al., 1993; U.S. application Ser. No. 08/131,625, now U.S. Pat. No. 5,695,766, and Ser. No. 08/301,435). ORFs 1a and 1b probably encode viral RNA polymerase (Meulenberg et al., 1993). ORFs 5, 6 and 7 were found to encode a glycosylated membrane protein (E), an unglycosylated membrane protein (M) and a nucleocapsid protein (N), respectively (Meulenberg et al., 1995). ORFs 2 to 4 appear to have the characteristics of membrane-associated proteins (Meulenberg et al., 1993; U.S. application Ser. No. 08/301,435). However, the translation products of ORFs 2 to 4 were not detected in virus-infected cell lysates or virions (Meulenberg et al., 1995).
The major envelope glycoprotein of EAV encoded by ORF 5 may be the virus attachment protein, and neutralizing monoclonal antibodies (MAbs) are directed to this protein (de Vries, J. Virol. 1992; 66:6294-6303; Faaberg, J. Virol. 1995; 69:613-617). The primary envelope glycoprotein of LDV, a closely related member of PRRSV, is also encoded by ORF 5, and several different neutralizing MAbs were found to specifically immunoprecipitate the ORF 5 protein (Cafruny et al., Vir. Res., 1986; 5:357-375). Therefore, it is likely that the major envelope protein of PRRSV encoded by ORF 5 may induce neutralizing antibodies against PRRSV.
Several hypervariable regions within the ORF5 were identified and were predicted to be antigenic (U.S. application Ser. Nos. 08/131,625 and 08/301,435). It has been proposed that antigenic variation of viruses is the result of direct selection of variants by the host immune responses (reviewed by Domingo et al., J. Gen. Virol. 1993, 74:2039-2045). Thus, these hypervariable regions are likely due to the host immune selection pressure and may explain the observed antigenic diversity among PRRSV isolates.
The M and N proteins of U.S. PRRSV isolates, including ISU 3927, are highly conserved (U.S. application Ser. No. 08/301,435). The M and N proteins are integral to preserving the structure of PRRSV virions, and the N protein may be under strict functional constraints. Therefore, it is unlikely either that (a) the M and N proteins are subjected to major antibody selection pressure or that (b) ORFs 6 and 7, which are likely to encode the M and N proteins, are responsible for or correlated to viral virulence. Interestingly, however, higher sequence variation of the LDV M protein was observed between LDV isolates with differing neurovirulence (Kuo et al., 1992, Vir. Res. 23:55-72).
ORFs 1a and 1b are predicted to translate into a single protein (viral polymerase) by frameshifting. ORFs 2 to 6 may encode the viral membrane associated proteins.
In addition to the genomic RNA, many animal viruses produce one or more sg mRNA species to allow expression of viral genes in a regulated fashion. In cells infected with PRRSV, seven species of virus-specific mRNAs representing a 3xe2x80x2-coterminal nested set are synthesized (mRNAs 1 to 7, in decreasing order of size). mRNA 1 represents the genomic mRNA. Each of the sg mRNAs contains a leader sequence derived from the 5xe2x80x2-end of the viral genome.
The numbers of the sg mRNAs differ among arteriviruses and even among different isolates of the same virus. A nested set of 6 sg mRNAs was detected in EAV-infected cells and European PRRSV-infected cells. However, a nested set of six (LDV-C) or seven (LDV-P) sg mRNAs, in addition to the genomic RNA, is present in LDV-infected cells. The additional sg mRNA 1-1 of LDV-P contains the 3xe2x80x2-end of ORF 1b and can potentially be translated to a protein which represents the C-terminal end of the viral polymerase. Sequence analysis of the sg mRNAs of LDV and EAV indicates that the leader-mRNA junction motif is conserved. Recently, the leader-mRNA junction sequences of the European LV were also shown to contain a common motif, UCAACC, or a highly similar sequence.
The sg mRNAs have been shown to be packaged into the virions in some coronaviruses, such as bovine coronavirus (BCV) and transmissible gastroenteritis virus (TGEV). However, only trace amounts of the sg mRNAs were detected in purified virions of mouse hepatitis virus (MHV), another coronavirus. The sg mRNAs of LDV, a closely related member of PRRSV, are also not packaged in the virions, and only the genomic RNA was detected in purified LDV virions.
The sg mRNAs of LDV and EAV have been characterized in detail. However, information regarding the sg mRNAs of PRRSV strains, especially the U.S. PRRSV, is very limited. Thus, a need is felt for a more thorough molecular characterization of the sg mRNAs of U.S. PRRSV.
The packaging signal of MHV is located in the 3xe2x80x2-end of ORF 1b, thus only the genomic RNA of MHV is packaged. The sg mRNAs of BCV and TGEV, however, are found in purified virions. The packaging signal of BCV and TGEV has not been determined. The Aura alphavirus sg mRNA is efficiently packaged into the virions, presumably because the packaging signal is present in the sg mRNA. The sindbis virus 26S sg mRNA is not packaged into virions because the packaging signal is located in the genome segment (not present in sg mRNA).
Several mechanisms are involved in the generation of the sg mRNAs. It has been proposed that coronaviruses utilize a unique leader RNA-primed transcription mechanism in which a leader RNA is transcribed from the 3xe2x80x2 end of the genome-sized negative-stranded template RNA, dissociates from the template, and then rejoins the template RNA at downstream intergenic regions to prime the transcription of sg mRNAs. The model predicts that the 5xe2x80x2-leader contains a specific sequence at its 3xe2x80x2-end which is repeated further downstream in the genome, preceding each of the ORFs 2 to 7. The leader joins to the body of each of the sg mRNAs via the leader-mRNA junction segment.
The various strains of PRRSV continue to be characterized (Halbur et al., J. Vet. Diagn. Invest. 8:11-20 (1996); Meng et al., J. Vet. Diagn. Invest. 8:374-381 (1996); Meng et al., J. Gen. Virol. 77:1265-1270 (1996); Meng et al., J. Gen. Virol. 76:3181-3188 (1995); Meng et al., Arch. Virol. 140:745-755 (1995); Halbur et al., Vet. Pathol. 32:200-204 (1995); Morozov et al., Arch. Virol. 140:1313-1319 (1995); Meng et al., J. Gen Virol. 75:1795-1801 (1994); Halbur et al., J. Vet. Diagn. Invest. 6:254-257 (1994), all of which are incorporated herein by reference in their entireties.)
aPRRSV is an important cause of pneumonia in nursery and weaned pigs. PRRSV causes significant economic losses from pneumonia in nursery pigs (the exact extent of which are not fully known). Reproductive disease was the predominant clinical outcome of PRRSV infections during the past few years, due to the early prevalence of relatively low virulence strains of PRRSV. Respiratory disease has now become the main problem associated with PRRSV, due to the increasing prevalence of relatively high virulence strains of PRRSV. A need is felt for a vaccine to protect against disease caused by the various strains of PRRSV.
Surprisingly, the market for animal vaccines in the U.S. and worldwide is larger than the market for human vaccines. Thus, there exists an economic incentive to develop new veterinary vaccines, in addition to the substantial public health benefit which is derived from protecting farm animals from disease.
Accordingly, it is an object of the present invention to provide a DNA sequence encoding a porcine reproductive and respiratory syndrome virus (PRRSV) which contains SEQ ID NO:55 (ISU-12) or SEQ ID NO:54 (ISU-55).
It is another object of the invention to provide a DNA sequence encoding an open reading frame of ISU-12 including nucleotides 191-7387 of SEQ ID NO:68 (ORF1a), nucleotides 7375-11757 of SEQ ID NO:69 (ORF 1b), nucleotides 11762-12529 of SEQ ID NO:70 (ORF 2), nucleotides 12385-13116 of SEQ ID NO:71 (ORF 3), nucleotides 12930-13463 of SEQ ID NO:72 (ORF 4), nucleotides 13477-14076 of SEQ ID NO:73 (ORF 5), nucleotides 14064-14585 of SEQ ID NO:74 (ORF 6) and nucleotides 14578-14946 of SEQ ID NO:75 (ORF 7);
or of ISU-55 of ISU-12 including nucleotides 191-7699 of SEQ ID NO:76 (ORF1a), nucleotides 7657-12009 of SEQ ID NO:77 (ORF 1b), nucleotides 12074-12841 of SEQ ID NO:78 (ORF 2), nucleotides 12697-13458 of SEQ ID NO:79 (ORF 3), nucleotides 13242-13775 of SEQ ID NO:80 (ORF 4), nucleotides 13789-14388 of SEQ ID NO:81 (ORF 5), nucleotides 14376-14897 of SEQ ID NO:82 (ORF 6) and nucleotides 14890-15258 of SEQ ID NO:83 (ORF 7).
It is also an object of the invention to provide a polypeptide encoded by the DNA sequence encoding ISU-12 or ISU-55, or one or more ORFs thereof.
Yet another object of the invention is to provide a composition for inducing antibodies against PRRSV comprising one or more polypeptides encoded by the DNA sequences of one or more ORF of ISU-12 or ISU-55.
Another object of the invention is to provide a method of protecting a pig from a porcine reproductive and respiratory disease, by administering an effective amount of the polypeptides encoded by the DNA sequences of one or more ORFs of ISU-12 or ISU-55 to a pig in need of protection against said disease.
It is yet another object of the invention to provide a method of distinguishing PRRSV strain ISU-55 from other strains of PRRSV by:
(a) amplifying a DNA sequence of the PRRSV using the following two primers.
55F 5xe2x80x2-CGTACGGCGATAGGGACACC-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:84)
and
3RFLP 5xe2x80x2-GGCATATATCATCACTGGCG-3xe2x80x2xe2x80x83xe2x80x83(SEQ ID NO:85);
(b) digesting the amplified sequence of step (a) with DraI; and
(c) correlating the presence of three restriction fragments of 626 bp, 187 bp and 135 bp with a PRRSV ISU-55 strain.
These and other objects, which will become apparent during the following description of the preferred embodiments, have been provided by a purified and/or isolated polypeptide selected from the group consisting of proteins encoded by one or more open reading frames (ORF""s) of an Iowa strain of porcine reproductive and respiratory syndrome virus (PRRSV), proteins at least 94% but less than 100% homologous with a protein encoded by an ORF 2 of an Iowa strain of PRRSV, proteins at least 88% but less than 100% homologous with a protein encoded by ORF 3 of an Iowa strain of PRRSV, proteins at least 93% homologous with an ORF 4 of an Iowa strain of PRRSV, proteins at least 90% homologous with an ORF 5 of an Iowa strain of PRRSV, proteins at least 97% but less than 100% homologous with proteins encoded by one or both of ORF 6 and ORF 7 of an Iowa strain of PRRSV, antigenic regions of such proteins which are at least 5 amino acids in length and which effectively stimulate protection in a porcine host against a subsequent challenge with a PRRSV isolate, and combinations thereof; an isolated polynucleic acid which encodes such a polypeptide or polypeptides; a vaccine comprising an effective amount of such a polynucleotide or polypeptide(s); antibodies which specifically bind to such a polynucleotide or polypeptide; methods of producing the same; and methods of (i) effectively protecting a pig against PRRS, (ii) treating a pig exposed to a PRRSV or suffering from PRRS, and (iii) detecting a PRRSV using the same.